The problem of infertility in animals caused by the abnormality and deficiency of sperms can be solved by Assisted Reproductive Technology (ART). However, the methods of ART require a high quality of sperms to enhance the fertilization rate. In this paper, we focused on investigating the factors that affect the quality of sperms, which were sorted by the simple design of microfluidic devices (MFD). The first factor was associated with the types of polymer, which is used for fabrication. The second factor was related to the distance that sperms swam. The last factor was the temperature that we used for incubating sperms. The results showed that the PDMS MFD was more effective than the SU-8 MFD, and the most suitable distance that the frozen-thawed bull sperms could swim with having less effect of sperm exhaustion was 5 mm. More importantly, we are the first to prove that incubating sperms in the MFD at 37 °C was unnecessary. The percentage of sperm viability for the room temperature (25 °C) was significantly higher than incubation at 37 °C. For this reason, it could be concluded that incubating sperms at 25 °C could be sufficient. This makes it possible to reduce the time consumption and cost of sorting sperms with the MFD as we can eliminate the incubating process and equipment. Moreover, we further assessed the quality of the sperms, which were sorted with PDMS MFD at the channel length 5 mm compared with the pre-sorted sperms. The results revealed that the percentage of progressive motile sperms significantly increased, while the percentage of abnormality significantly reduced. Based on these results, it could be concluded that sorting sperm with a PDMS MFD has a less negative effect on the quality of the sperms.